An In-Depth Glycosylation Assay for Urinary Prostate-Specific Antigen

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An In-depth Glycosylation Assay for Urinary Prostate Specific Antigen Guinevere Stevina Margaretha Kammeijer, Jan Nouta, Jean de la Rosette, Theo M. de Reijke, and Manfred Wuhrer Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04281 • Publication Date (Web): 04 Mar 2018 Downloaded from http://pubs.acs.org on March 6, 2018

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Analytical Chemistry

1

An In-depth Glycosylation Assay for Urinary Prostate

2

Specific Antigen

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Guinevere S.M. Kammeijer1*, Jan Nouta1, Jean J.M.C.H. de la Rosette2, Theo M. de Reijke3, Manfred

4

Wuhrer1

5

1

Leiden University Medical Center, Center for Proteomics and Metabolomics, Leiden, The Netherlands

6

2

Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

7

3

Academic Medical Center, Department of Urology, Amsterdam, The Netherlands

8

*

9

Proteomics

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Correspondence: Guinevere S.M. Kammeijer, Leiden University Medical Center, Center for and

Metabolomics,

P.O.

Box

9600,

2300

RC

Leiden,

The

Netherlands;

[email protected]; Tel: +31-71-52-69384

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ABSTRACT – MAX. 250 WORDS

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The concentration of prostate specific antigen (PSA) in serum is used as an early detection method of

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prostate cancer (PCa) showing, however, low sensitivity, specificity and a poor predictive value. Initial

14

studies suggested the glycosylation of PSA to be a promising marker for a more specific yet non-

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invasive PCa diagnosis. Recent studies on the molecular features of PSA glycosylation (such as antenna

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modification and core fucosylation) were not successful in demonstrating its potential for an improved

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PCa diagnosis, probably due to the lack of analytical sensitivity and specificity of the applied assays. In

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this study, we established for the first time a high-performance PSA Glycomics Assay (PGA), allowing

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differentiation of α2,6- and α2,3-sialylated isomers, the latter one being suggested to be a hallmark of

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aggressive types of cancer. After affinity purification from urine and tryptic digestion, PSA samples

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were analyzed by CE-ESI-MS (capillary electrophoresis – electrospray ionization coupled to mass

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spectrometry). Based on positive controls, an average inter-day relative standard deviation of 14% for

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41 N-glycopeptides was found. The assay was further verified by analyzing PSA captured from

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patients’ urine samples. A total of 67 N-glycopeptides were identified from the PSA pooled from the

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patients. In summary, the first PGA successfully established in this study allows an in-depth relative

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quantitation of PSA glycoforms from urine. The PGA is a promising tool for the determination of

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potential glycomic biomarkers for the differentiation between aggressive PCa, indolent PCa and

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benign prostate hyperplasia in larger cohort studies.


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1.

INTRODUCTION

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Since 1994, the protein concentration of prostate specific antigen (PSA) in serum is a FDA-approved

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early detection method for prostate cancer (PCa), although its sensitivity is reportedly rather poor.1

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When the PSA value is found to be elevated (above 3 ng/mL or 4 ng/mL), the patient will be advised to

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undergo more thorough investigations (e.g. digital rectal examination (DRE), multiparametric magnetic

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resonance imaging (mpMRI) and/or prostate biopsy).1,2 Additional examinations are mandatory since

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the PSA test lacks the specificity that is required for the early diagnosis of PCa, as age, benign prostate

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hyperplasia (BPH) or prostatitis can also result in elevated levels of PSA.1 Furthermore, a recent study

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by Foley et al. showed that the PSA test has an overall low predictive value for PCa and is incapable of

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discriminating between aggressive and non-aggressive prostate tumors.3,4 Therefore, the need for

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more specific and predictive biomarkers is eminent, to effectively discriminate between aggressive

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and non-aggressive tumors and to avoid unnecessary prostate biopsies.

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Literature suggests that the analysis of PSA glycosylation could offer a more efficient PCa diagnosis,5-8

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since the single N-linked glycosylation site (N69) of PSA is occupied by a very heterogeneous group of

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glycans.5-7 Differences in the linkages of sialic acids, which can be α2,3-, α2,6- or α2,8-linked, have

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been associated with cancer progression,9 with α2,6-sialylated glycans involved in blocking the galectin

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binding to β-galactoside, and α2,3- sialylated glycans suggested to be involved in (aggressive) types of

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cancer.10,11 Further investigations into whether a higher abundance of α2,3-sialylated species present

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on PSA could be correlated to PCa, found an overall a higher specificity and sensitivity when compared

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to the conventional PSA test.12 However, this study disregarded the presence of α2,6-sialylated glycans

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and did not detect other molecular features such as fucosylation or bisection. Another recent study

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investigated the glycosylation features of PSA in urinary samples collected after DRE by using an

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immunopurification step.13 Lectins were used to investigate the level of core fucosylation and the

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presence of α2,6-linked sialic acids, revealing, however, no association with the pathology of the

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disease (Gleason score). This observation could result from the rather small sample size (18 controls vs

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36 PCa cases) and the only partial cover of sialic acid linkages, since only the levels of α2,6-sialic acids

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were considered. Another study compared the total serum N-glycome of PCa patients and BPH

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patients, revealing differences with respect to sialylation as well as fucosylation.7 An overall increase

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of α2,3-sialylated glycans and alterations in fucosylation was shown in PCa patients, which is in

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agreement with previous studies.14,15 Notably, as these studies analyzed the total plasma or serum N-

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glycome after glycan release, no information was obtained regarding the protein carriers of the

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affected glycans. Several other groups explored specifically the level of fucosylation of serum PSA,

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demonstrating an overall increase in total fucosylation for free PSA16 or total PSA,17 whilst another

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study reported decreased N-glycan core-fucosylation and an increase in α2,3-sialylated glycans for PSA Page 3 of 19 ACS Paragon Plus Environment


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in PCa patients.18 In a previous study by CE-ESI-MS (capillary electrophoresis – electrospray ionization

64

coupled to mass spectrometry), on a proteolytically cleaved PSA standard (human seminal plasma) we

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could identify 75 different glycan species (including isomeric separation of differently linked sialic

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acids).19 Additionally, a recent in depth-study by liquid chromatography – electrospray ionization

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coupled to mass spectrometry (LC-ESI-MS(/MS)) on a similar sample, identified 23 glycan compositions

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on the single N-glycosylation site of PSA and in addition showed, with a relatively small cohort (61 BPH

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and 31 PCa cases), that for urinary PSA the fucosylation was decreased in PCa patients and the level of

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sialylation was increased compared to BPH patients.20 While some of these studies noted differences

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in the level of PSA sialylation and fucosylation, they often featured limited precision, sensitivity and/or

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resolution.9,16-18,14,20

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Answering to the need for an analytically more specific and sensitive high-resolution assay to assess

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PSA glycosylation features of benign, indolent or aggressive tumors, we report here on an in-depth

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high-performance urinary PSA Glycomics Assay (PGA) for patients suspected of PCa, combining the

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strengths of previously published assays. The assay includes the capturing of PSA from urine, an in-

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solution digestion, and the analysis of glycopeptides with CE-ESI-MS(/MS), taking advantage of the

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separation of sialylated isomers in CE. In addition, as restricted quantities of PSA are found in urine

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(before DRE),15 a sheathless interface (CESI) with a dopant enriched nitrogen gas was used, improving

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analytical sensitivity and repeatability for glycopeptide analysis,21 making this assay suitable to detect

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rather small differences in the relative abundance of different glycoforms.

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2.

EXPERIMENTAL SECTION

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2.1.

MATERIALS

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Ammonium bicarbonate (ABC), ethanol (EtOH), sodium bicarbonate (NaHCO3), sodium chloride (NaCl),

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sodium hydroxide (NaOH), sodium phosphate dibasic dihydrate (Na2HPO4∙2H2O) and monopotassium

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phosphate (KH2PO4) were obtained from Merck (Darmstadt, Germany). Glacial acetic acid, DL-

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dithiothreitol (DTT), hydrochloric acid (HCl) and iodoacetamide were acquired from Sigma-Aldrich

88

(Steinheim, Germany). Ammonium acetate, formic acid (FA) and water of LC-MS grade water were

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purchased from Fluka (Steinheim, Germany). Trypsin from bovine pancreas was obtained from

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Promega (Madison, WI). Milli-Q water (MQ) was obtained using a Q-Gard 2 system (Millipore,

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Amsterdam, The Netherlands). HPLC supraGradient acetonitrile (MeCN) was acquired from Biosolve

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(Valkenswaard, The Netherlands). PSA standard derived from semen was purchased from Lee

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BioSolutions (St. Louis, MO). Five times concentrated (5x) PBS consisted out of 0.16 M Na2HPO4, 0.02


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M KH2PO4, 0.73 M NaCl at pH 7.2. 1xPBS was prepared from the 5xPBS by diluting it with MQ and

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resulting in a pH of 7.6.

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2.2.

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Urine samples from 10 healthy female volunteers were collected at the Leiden University Medical

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Center. As a negative control, a female urine pool (FUP) was made by pooling 10 urine portions. One

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urine sample from a healthy male volunteer was collected at the Academic Medical Center

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(Amsterdam, The Netherlands) as well 25 urine samples from patients suspected of PCa. All patients

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had a serum PSA level of >3 ng/mL and donated their urine prior to prostate biopsy and before DRE.

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Clinical information and urine specimens were collected with the approval of the medical ethical

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committee of the AMC (W16_010#16.020). Table 1 illustrates the clinical information of the patients.

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Urine was collected (8 -72 mL) and cooled down to room temperature (RT) before storing it at -80˚C.

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2.3.

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Custom-made anti-PSA specific nanobodies based on sequence N7 from Saerens et al.22 (antigen

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binding portion of the heavy chain from camelids, 0.49 mg/mL, PBS) were obtained from QVQ

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(Utrecht, The Netherlands) and coupled to NHS activated Sepharose 4 Fast Flow-beads (GE Healthcare,

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Little Chalfont, United Kingdom) according to the manufacturer’s protocol. Briefly, 7 mL 0.49 mg/mL

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anti-PSA in PBS was added to 14 mL drained beads (1:1200 molar ratio; anti-PSA:NHS), the mixture

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was incubated for 2 hours at RT, the solution was spun down, supernatant was removed and the

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beads were resuspended in 50 mL of 0.1 M Tris-HCl pH 8.5 and incubated for 2 hours at RT.

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Immobilization of the nanobodies was confirmed by analyzing the supernatant on a NuPAGE® SDS-

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PAGE gel (Thermo Fisher, Waltham, MA) with NuPAGE® MOPS SDS running buffer (Thermo Fisher) and

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after staining with Coomassie G-250 (SimplyBlue SafeStain, Colloidal Blue Staining Kit, Thermo Fisher).

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Antibody beads were stored as a 50% bead suspension (v/v) in 20% EtOH (20:80, EtOH:H2O, v/v) at

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4˚C. Before using the beads, the 50% bead suspension was washed with 1xPBS for ethanol removal

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and re-suspended in 1xPBS to produce a 50% bead suspension (v/v).

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2.4.

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After thawing the urine samples to RT, cell debris and other particulates were precipitated by

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centrifugation (500 g, 5 min) and removed from the supernatant. Almost all samples contained 20 mL

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urine, in case 20 mL was not available, MQ was added to the urine sample to obtain a total volume of

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20 mL. For the intra- and inter-day variation and as positive control for the cohort analysis, a volume

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of 20 mL of the FUP was spiked with 15 µL of a 0.1 mg/mL PSA standard derived from semen.

CLINICAL SAMPLES

ANTI-PSA BEADS

PSA CAPTURING



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For the capturing procedure, several parameters were tested such as, amount of bead suspension,

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potential non-specific binding of the beads, material of the retainer during capturing, pH of the

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sample, influence of a blocking agent and whether the elution would be hampered if the beads ran dry

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during the washing procedure. In order to determine the optimal amount of beads, we added

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different volumes of 50% anti-PSA bead suspension to 5 mL of five times concentrated (5x)PBS and 20

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mL urine. In addition, we tested whether NHS beads showed non-specific binding for PSA. For this, we

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added bare, inactivated NHS beads to a FUP spiked with a PSA standard. Next, we tested the material

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of the retainer used for the capturing procedure comparing plastic (Eppendorf tubes, Hamburg,

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Germany) with glass (Grace, Columbia, MD). This was combined with the testing of different volumes

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of spiked FUP (150 µL, 1000 µL, 2000 µL and 5000 µL). Furthermore, we tested whether the pH of the

135

FUP influenced the capturing efficiency. This was performed by adjusting the pH of the FUP to pH 6.0,

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7.0 and 7.8 using PBS at pH 6, pH 7 and a TRIS/NaCl solution to achieve pH 8. We then evaluated the

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addition of a blocking agent (bovine serum albumin (BSA) or casein) to the capturing mixture in order

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to limit possible non-specific binding of PSA to the retainer. Finally, the incubation time (2, 4, 6, 8 and

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23 hrs) as well as the temperature (4˚C vs RT) were varied with incubation of the sample on a tube

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roller. For the capturing of PSA from patient material the following final parameters were used: 60 µL

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of 50% anti-PSA bead suspension, 20 mL plastic Falcon tube, urine adjusted to pH 7, no blocking agent

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and an overnight incubation at 4˚C. After capturing, the sample was centrifuged at 100 g for 1 min at

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RT. After removal of the supernatant, the remaining anti-PSA bead suspension (roughly 500 µL) was

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transferred to a 96-well polypropylene filter plate (2 mL) containing a 10 µm pore size polyethylene

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frit (Orochem, Naperville, IL). The liquid was further removed by using a vacuum manifold

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(MerckMillipore, Darmstadt, Germany). Finally, it was tested whether the elution was hampered if the

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beads were completely dried (one minute of extra vacuum was applied) during the washing with 600

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µL 1xPBS (pH 7.6) followed by two times 600 µL 50 mM ABC. After this step, PSA was eluted by adding

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200 µL of 100 mM FA that was collected in a V-bottom microtiter plate; the sample was evaporated at

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45˚C in a vacuum centrifuge (Eppendorf Concentrator 5301, Eppendorf) and reconstituted in 5 µL of

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25 mM sodium bicarbonate.

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2.5.

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For all SDS-PAGE experiments, samples were separated by NuPAGE® 4-12% gradient Bis-Tris gel

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(Thermo Fisher). For reduction, 2.5% of 2-mercaptoethanol (Sigma-Aldrich) was added to the sample

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prior to SDS-PAGE. The gel was stained with colloid blue (Novex). For in-gel digestions the PSA bands

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(~32 kDa) were excised and cut into pieces that were washed with 100 µL 25 mM ABC with an

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incubation time of 5 min at RT. Supernatant was removed and the gel pieces were dehydrated by the

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addition of 100 µL 30% MeCN with an incubation time of 30 min at RT, after which the supernatant

SDS-PAGE AND IN-GEL TRYPTIC DIGESTION


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was removed. A final volume of 100 µL 10% MeCN was added with an incubation time of 10 min at RT

160

prior to reduction with 75 µL of 0.9 mM DTT for 30 min at 56˚C. After reduction the supernatant was

161

removed and 100% MeCN was added for 10 min at RT. After removal of the supernatant, 75 µL of 5

162

mM iodoacetamide was added and kept in the dark for 20 min at RT. The supernatant was removed

163

after the alkylation and 100 µL of 100% MeCN was added for 5 min at RT. After removal, 25 mM ABC

164

was added and the gel pieces were incubated for 10 min at RT. The supernatant was removed and an

165

additional 100% MeCN was added and incubated at RT for 5 min. After removal of the supernatant,

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the gel pieces were dried in a vacuum centrifuge for 30 min. Samples were reconstituted in a mixture

167

containing the proteolytic enzyme (30 µL of 5 ng/µL trypsin in 25 mM ABC). Digestion was performed

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overnight at 37˚C. The capturing efficiency was determined using software package Gelanalyzer 2010.

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The software was used to quantify the capturing efficiency by comparing the intensities of the PSA

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protein bands in SDS-PAGE gel (.png file) before and after capturing.

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2.6.

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The 5 µL reconstituted eluates, described in section 2.4, were reduced and alkylated by adding 1 µL of

173

12 mM DTT with an incubation time of 30 min at 60˚C followed by the addition of 1 µL 42 mM

174

iodoacetamide with an incubation of 30 min performed in the dark at RT. To ensure that the sulfide

175

alkylation was stopped, 1 µL of 48 mM DTT was added and incubated for 20 min in full light at RT.

176

After addition of 1 µL of 0.15 mg/mL trypsin in 25 mM ABC, the digestion was performed overnight at

177

37˚C.

178

2.7.

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All experiments were performed under optimized conditions previously established.21 Briefly, a 90 cm

180

long bare-fused capillary (30 μm internal diameter and 150 μm outer diameter) was used on a CESI

181

8000 system (Sciex, Framingham, MA). The system had a temperature controlled sampling tray and for

182

all experiments a voltage of 20 kV was applied. Prior to each analysis, the separation capillary was

183

thoroughly rinsed with 0.1 M NaOH (2.5 min), LC-MS grade water (3 min), 0.1 M HCl (2.5 min), water

184

(3 min) and 3 min with the background electrolyte (BGE) of 10% acetic acid (v/v, 1.74 M, pH 2.3). The

185

conductive liquid line was supplemented with BGE, by rinsing with BGE for 3 min. An online pre-

186

concentration step was applied, transient isotachophoresis, by adding 1.0 μL of the leading electrolyte

187

(LE, 250 mM ammonium acetate at pH 4.0) to 1.5 μL of the sample. All samples were injected hydro-

188

dynamically by applying 1 psi pressure for 60 s, corresponding to 1.4% of the total capillary volume (9

189

nL). After each sample injection, a BGE postplug was injected by applying 0.5 psi for 25 s (0.3% of the

190

capillary volume).

IN-SOLUTION TRYPTIC DIGESTION

CAPILLARY ELECTROPHORESIS – ELECTROSPRAY IONIZATION–MASS SPECTROMETRY



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The CE system was coupled to a UHR-QqTOF maXis Impact HD MS (Bruker Daltonics, Bremen,

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Germany) via a sheathless CE-ESI-MS interface (SCIEX). The complete housing of the nozzle was a

193

customized platform from SCIEX, which allowed the optimal alignment of the position of the capillary

194

spray tip in front of the nanospray shield (Bruker Daltonics). A stable electrospray was obtained by

195

applying a glass capillary voltage between -1100V and -1300V. All experiments were performed in

196

positive ionization mode. The flow rate and temperature of the drying gas (nitrogen) were adjusted to

197

1.2 L/min and 150°C, respectively. MS data were acquired between m/z 200 and 2200 with a 1 Hz

198

spectral acquisition frequency. An internal polymer cone was attached onto the porous tip housing to

199

enable the usage of a dopant enriched nitrogen (DEN) gas with MeCN as a dopant (ca. 4%, mole

200

percent).21

201

2.8.

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CE-ESI-MS data was analyzed with Data Analysis 4.2 (Build 395, Bruker Daltonics). Calibration of the

203

MS spectra was performed prior to data analysis with sodium adducts that were present at the

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beginning of the electropherogram. The data was manually screened for glycopeptides based on the

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exact mass, migration order and relative intensities (Supporting Information, Table S-1).

206

Fragmentation spectra were acquired for 30% of the identified compositional glycopeptide variants of

207

PSA (Supporting Information, Figures S-1A – S-1T), an additional 16% was identified on the basis of

208

previous research.19 Extracted ion electropherograms (EIEs, smoothed with a Gaussian fit) were

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acquired with the first three isotopes of the double, triple and quaternary charged analytes using a

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width of ± m/z 0.05 unit.

DATA ANALYSIS


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3. RESULTS AND DISCUSSION

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A PSA Glycomics Assay (PGA) for the glycosylation analysis of PSA from urine of patients suspected of

213

PCa was established and verified (ܰ = 25). For this, PSA was captured from typically 20 mL of urine,

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subjected to tryptic treatment, and the obtained glycopeptides were analysed by CE-ESI-MS. For data

215

processing, glycopeptide signals were integrated, and ratios of the glycopeptide signal intensities were

216

determined. The PGA was further verified by examining factors such as capturing procedure,

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repeatability, intermediate precision and biological variation between patients.

218

3.1.

219

Several parameters of the capturing procedure were optimized and are summarized in Supporting

220

Information, Table S-2. Briefly, a volume of 60 µL of anti-PSA beads was found to give the highest

221

capturing efficiency (N = 2, Supporting Information, Figure S-2). The use of bare NHS Sepharose beads

222

did not result in non-specific binding of PSA as the PSA was observed only in the flow-through and not

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in the eluate of the bare NHS Sepharose beads, while anti-PSA beads did capture PSA and no PSA was

224

observed in the flow through (Supporting Information, Figure S-3). PSA recoveries were largely

225

comparable when using plastic vs. glass retainers, and plastic retainers were chosen for their

226

convenience for further experiments (N = 1, Supporting Information, Figure S-4). Furthermore, the pH

227

of the FUP (N = 2), was investigated and a similar recovery was found for all conditions (pH 6, 7 and

228

8) (Supporting Information, Figure S-5). For subsequent experiments pH 7 was chosen. It was tested

229

whether the addition of a blocking agent prevented nonspecific binding of PSA to the walls of the

230

retainer and could improve the capturing efficiency (Supporting Information, Figure S-6). Notably, the

231

use of bovine serum albumin (BSA) and casein as a blocking agent contaminated the affinity-purified

232

sample, but did not result in increased PSA yields. Therefore, for further experiments no blocking

233

agent was added to the FUP. Incubation at room temperature showed a wide variation in capturing

234

efficiency, with 4˚C being the most preferable temperature (Supporting Information, Figure S-7). The

235

incubation time did not appear to influence the PSA recovery, as a recovery between 72% and 78%

236

was observed across the range of incubation times (2, 4, 6, 8 hrs and overnight, N = 1). Due to

237

practical considerations, further experiments were performed with an overnight incubation. As a

238

vacuum system was used with a 96-well format for the washing and elution of the PSA from the anti-

239

PSA beads, it was tested whether PSA yields changed when the 96-well plate was run dry during the

240

washing procedure (an extra minute of vacuum was applied, Supporting Information, Figure S-8). A

241

similar capturing efficiency was observed when the well was completely dried compared to wells that

242

were kept wet during the washing procedure (80% vs 73%, N = 2). FA (100 mM) proved to be a

243

suitable elution reagent, as no PSA was detected post-elution on the beads after the beads were

244

incubated with the loading buffer and analyzed on a NuPAGE® SDS-PAGE gel (data not shown).

CAPTURING PROCEDURE



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Furthermore, PSA quantitation by SDS-PAGE with Coomassie staining ( N = 3 , Supplementary

246

Information, Figure S-9) revealed that the capturing procedure recovered approximately 58% of the

247

PSA spiked into a female urine pool (FUP). To reveal any potential glycosylation bias of the affinity

248

capturing procedure, glycosylation profiles of PSA before and after capturing were compared, applying

249

in-gel tryptic cleavage of PSA followed by CE-ESI-MS. We chose for a gel-based approach in order to

250

remove any possible sample matrix confounders. A total of 41 N-glycopeptides were detected, and

251

comparable profiles were obtained before and after affinity purification ( ܰ = 3, Supporting

252

Information, Figure S-10). The relative abundances of the 10 most abundant N-glycopeptides are

253

compared in Figure 1 and show no bias of the glycosylation profile induced by the affinity purification

254

procedure, with an average relative standard deviation (RSD) of 6.5% (non-captured) and 3.0%

255

(captured) for the ten most abundant glycopeptides (normalized to the sum of all identified

256

glycoforms). In addition, an average RSD of 14.8% (non-captured) and 8.3% (captured) for all

257

glycopeptides was observed. However, it has to be noted that the in-gel digestion was performed on

258

the ~32 kDa band and possible discrimination of potential nicked forms of PSA that may migrate at

259

different heights is, therefore, not taken into account.

260

3.2.

261

The analysis of a tryptic digest of the captured PSA standard identified 41 N-glycopeptides with the CE-

262

ESI-MS set-up. All glycans were attached to the dipeptide N69K as listed in Supporting Information,

263

Table S-1. The base peak electropherogram of a typical PSA tryptic digest analysis (Figure 2) shows

264

three distinctive glycopeptide clusters separated on the basis of the level of sialylation (Figures 2B and

265

2C). Furthermore, isomer separation was achieved for α2,3- and α2,6-sialylated species, in accordance

266

with our previous work.19 Figures 2B and 2C demonstrate that α2,3- and α2,6-linked isomers are

267

baseline resolved, confirming the ability of CE-ESI-MS to discriminate between the different linkages.

268

However, compared to our previous work,19 non-sialylated glycoforms were not detected in this

269

sample. The difference is most likely due to the use of different PSA batches in the two studies.

270

Notably, the peaks that are observed in the base peak electropherogram in the non-sialylated region

271

(area marked by an asterisk in Figure 2A) correspond to other analytes in the sample such as tryptic

272

unglycosylated peptides.

273

3.3.

274

Since SDS-PAGE demonstrated sufficient purity of the affinity-enriched PSA, an in-solution proteolytic

275

cleavage was chosen for the PGA. For the precision evaluation of the assay 20 mL FUP was spiked with

276

1.5 µg of a PSA standard and the complete assay (including capturing) was executed three times on

277

the same day (repeatability, intra-day variability) and repeated over three days (intermediate

PSA GLYCOPEPTIDE DETECTION BY CE-ESI-MS

REPEATABILITY AND INTERMEDIATE PRECISION


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precision, inter-day variability). As illustrated in Figure 3, the average intra-day and inter-day RSD were

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below 3% and 7%, respectively, for the ten most abundant glycopeptides (normalized to the sum of all

280

identified glycoforms). Results for the remaining 31 identified N-glycopeptides (relative abundance of

281

low abundant glycopeptides) are shown in Supporting Information, Figure S-11. The RSDs for all

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glycopeptides showed an average intraday variation of 5% and an average inter-day variation of 14%.

283

3.4.

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The biological variation in PSA glycosylation was examined using the developed PGA applied to urine

285

samples of 25 patients. Before sample treatment, the patient urine samples were split into three

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batches, each of them processed on one of three consecutive days. Each batch included a negative

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(FUP) and a positive control (FUP spiked with 1.5 µg of standard PSA). Furthermore, after tryptic

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digest, a small portion (0.5 µL) of all the patient samples was pooled. This pool was used for the

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identification of the glycopeptides and structural elucidation by tandem MS (ܰ = 3). A total of 67 N-

290

glycopeptides could be identified in the pooled patient sample. All glycans were attached to the

291

dipeptide N69K, in agreement with the previous result, and a complete overview of the glycopeptides

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is given in Supporting Information, Table S-1. After data treatment, 23 of the 25 patient samples

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passed the quality criteria (>20 glycopeptides identified with S/N >9, ppm error

An In-Depth Glycosylation Assay for Urinary Prostate-Specific Antigen

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